Skeletal disorders such as osteoarthritis and bone/cartilage wounds are currently difficult or impossible to treat. The ability to generate specific skeletal progenitor cells may provide the basis for new treatment therapies using a regenerative medicine approach. In order for this to occur it is essential to understand how skeletal progenitors arise during development. Appendicular skeletal progenitors, in particular, are generated when lateral plate mesoderm undergoes an epithelial-to- mesenchymal transition to generate mesenchymal skeletal progenitors that migrate away from the body axis to form the limb buds. Deciphering the mechanism that stimulates the generation of limb skeletal progenitors is important for understanding and treating appendicular skeletal defects. Recent studies have shown that retinoic acid (RA), an active metabolite of vitamin A, is required specifically for generation of forelimb skeletal progenitors: studies on RA-deficient mutant mouse embryos demonstrated a blockage of forelimb but not hindlimb budding. RA signaling is controlled by RA-synthesizing enzymes, including retinol dehydrogenase 10 (RDH10) that metabolizes retinol (vitamin A) to retinaldehyde, followed by retinaldehyde dehydrogenase 2 (RALDH2; ALDH1A2) that metabolizes retinaldehyde to RA. Interestingly, a forelimb-specific role for RA was shown by a recent genome-wide association study showing that severe osteoarthritis of the hand (the most common form of arthritis), but not hip or knee osteoarthritis, is associated with human ALDH1A2 gene variants that exhibit lower expression in articular cartilage. RA directly regulates gene transcription by functioning as a ligand for nuclear RA receptors that bind RA response elements near target genes. During development, Raldh2-/- embryos (that lack RA activity) fail to induce expression of Tbx5, the earliest known marker of forelimb development, whereas Rdh10-/- embryos (with greatly reduced RA activity) exhibit delayed Tbx5 expression limited to a smaller domain. Preliminary studies suggest that one mode of RA action may be repression of trunk FGF signaling to permit onset of Tbx5 expression. However, alternative mechanisms of RA action may exist including direct RA regulation of Tbx5 or RA regulation of Hox4/5 genes that may be required for induction of Tbx5 in the forelimb field. We will use a genetic loss-of-function approach to uncover the mechanism through which RA interacts with Tbx5, Hox genes, and FGF signaling to stimulate differentiation of lateral plate mesoderm to a forelimb skeletal progenitor fate. This project will generate important basic information on the signaling mechanisms needed for generation of appendicular skeletal progenitors that may aid in the treatment of skeletal disorders.